The overall goal of this proposal is to understand the in vivo metal ion selectivity for eukaryotic manganese superoxide dismutase (SOD2). SOD2 is a critical anti-oxidant enzyme of the mitochondria. The enzyme is active when manganese is bound to the catalytic site, but in vivo interactions with other metals such as iron have not been explored. We have previously shown that SOD2 activity is impaired in mtm1delta mutant strains of yeast that accumulate high mitochondrial iron. We will now test whether iron can inhibit SOD2 activity in vivo and whether Mtm1p facilitates selectivity for the correct metal. We shall (Aim 1) determine whether iron can occupy the metal binding site of SOD2 and inactivate the enzyme in vivo. The activity of SOD2 and metal content of the enzyme purified to homogeneity will be assessed as a function of intracellular manganese and iron availability. Secondly (Aim 2), we shall test whether the high mitochondrial iron ofmtml mutants is responsible for loss of SOD2 activity by genetically and nutritionally controlling intracellular iron levels. In Aim 3, transcriptional genome-wide microarray analysis will be utilized to determine the complex cellular response to loss of Mtmlp and pathways that lead to inactivation of SOD2. Together, these studies are designed to advance our understanding of how eukaryotic SOD2 obtains its manganese co-factor in vivo.